Human idiopathic pulmonary fibrosis (IPF) is a progressive, lethal fibrotic lung disease characterized by non-resolving epithelial injury, persistent myofibroblast (MFB) phenotype and stiffening of the extracellular matrix (ECM). Biomechanical signals derived from stiff/fibrotic ECM are emerging as crucial factors that regulate fibrotic lung progression. In recent studies, we have demonstrated that RhoA/Rho kinase (ROCK) mediate matrix stiffness sensing by lung MFBs. Inhibition of RhoA/ROCK mechanosensitive signaling ameliorates experimental lung fibrosis. MFBs isolated from patients with IPF are characterized by an invasive phenotype. It is currently not known whether pathologic ECM-derived biomechanical signaling regulates the invasive phenotype of IPF MFBs. Preliminary studies showed that matrix stiffness regulates IPF MFB invasion into the basement membrane (BM) by a RhoA/ROCK-dependent mechanism. Stiff matrix upregulates gene expression of integrin alpha 6 (ITGA6), matrix metalloproteinase 9 (MMP9) and urokinase-type plasminogen activator receptor (uPAR), factors associated with BM binding and degradation. Blocking ITGA6-mediated cell adhesion abrogates stiff matrix-dependent MFB invasion into the BM. Stiff matrix promotes ROCK-dependent phosphorylation of c-Fos and c-Jun, components of activator protein (AP-1) transcription factor complex, and selectively increases c-Fos and c-Jun binding to immobilized oligonucleotides containing AP-1-binding elements (TREs). Bioinformatics identified multiple TREs in the promoter regions of ITGA6, MMP9 and uPAR. These findings suggest that stiff matrix-induced RhoA/ROCK mechanosensitive signaling promotes MFB invasion into the BM by AP-1-dependent activation of an invasive gene program involving ITGA6, MMP9 and uPAR. The preliminary studies together with our previous studies suggest that RhoA/ROCK mechanosensitive signaling activates multiple fibrogenic mechanotransduction pathways through which sustained RhoA/ROCK signaling amplifies epithelial injury-induced lung fibrosis. In this project, we hypothesize that stiff matrix- induced RhoA/ROCK mechanosensitive signaling regulates MFB invasion into the BM and promotes persistent/progressive lung fibrosis. Specific aims are to: (1) determine whether c-Fos and c-Jun of AP-1 transcription factor complex mediate RhoA/ROCK mechanosensitive signaling to activate invasive gene program; (2) determine whether ITGA6, MMP9 and/or uPAR mediate RhoA/ROCK mechanosensitive signaling to regulate MFB invasion into the BM; and (3) determine whether sustained RhoA/ROCK mechanosensitive signaling following bleomycin-induced lung injury promotes persistent/progressive lung fibrosis in mice. The proposed study, if proven, will provide novel mechanistic insights into the promulgation of invasive MFB phenotype. It will provide proof-of-concept for pathologic ECM-derived biomechanical signaling in the pathogenesis of persistent/progressive fibrosis. The long-term goal of this project is to understand the biomechanical signal mechanisms of lung fibrosis and identify novel targets for effective anti-fibrotic therapies.